Direct recognition of homology between double helices of DNA in Neurospora crassa.

Abstract

Chromosomal regions of identical or nearly identical DNA sequence can preferentially associate with one another in the apparent absence of DNA breakage. Molecular mechanism(s) underlying such homology-dependent pairing phenomena remain(s) unknown. Using Neurospora crassa repeat-induced point mutation (RIP) as a model system, we show that a pair of DNA segments can be recognized as homologous, if they share triplets of base pairs arrayed with the matching periodicity of 11 or 12 base pairs. This pattern suggests direct interactions between slightly underwound co-aligned DNA duplexes engaging once per turn and over many consecutive turns. The process occurs in the absence of MEI3, the only RAD51/DMC1 protein in N. crassa, demonstrating independence from the canonical homology recognition pathway. A new perspective is thus provided for further analysis of the breakage-independent recognition of homology that underlies RIP and, potentially, other processes where sequence-specific pairing of intact chromosomes is involved.

a, RIP is quantified by the number of mutations in a random sample of germinated spores. RIP substrates are introduced into haploid nuclei of Strain A (filled circles, orange). Maternal homokaryotic tissues originate from wild-type Strain B (filled circles, magenta). RIP occurs in heterokaryotic cells (outlined in red) containing nuclei of both parental strains. The heterokaryotic cell and the nuclei are not drawn to scale.b, Direct perfect repeats of graded length assayed for RIP in c, d, e. The total sequenced region and the longest sub-region that is invariant among all constructs are indicated.c, The total number of mutated DNA strands for repeat constructs in b. 48 strands (24 spores from a single cross) were analyzed for each construct ().d, The total number of mutations in the invariant region (green) and its two sub-regions: 802-bp (blue, some or all of which is part of the repeat unit) and the 706-bp (grey, linker). Regression analysis is based only on mutation counts over the whole invariant region and includes the medium (524-220 bp) repeat range. r represents Pearson's correlation coefficient.e, Mutation profiles of selected repeat constructs.

424 mutations (99 mutated strands) were found in 150 sequenced spores.a, Spores were classified by the number of mutations over the total sequenced region. The number of spores (histogram) and the number of mutations (pie chart) per each class are shown. If the distribution of mutation counts followed a Poisson model (expected under the null assumption of independence of individual mutation events), the variance-to-mean ratio (VMR, distributed as the reduced chi-square under the Poisson model), would be equal to 1. VMR was found to be 30.98, significantly larger than 1 (P < 1*10-6), indicating strong over-dispersion of mutation counts.b, Mutation profile summarizes C/T and G/A mutations in 150 sequenced spores including the 24 spores analyzed earlier (). To determine if ample mutation of the linker region was associated with accidental bursts of RIP activity, every mutation was assigned two attributes: [1] its location in the construct, being either in the repeat unit, or the linker, or the flanking region, and [2] the total number of mutations found together on the same stand. The data did not reveal any relationship between the number of mutations on a given strand and their partitioning into single-copy or repetitive regions (P = 0.11, Fisher's Exact Test), suggesting that accidental bursts of RIP activity did not preferentially affect the linker region.

a, The general structure of repeat constructs analyzed in b-f. Each bipartite repeat unit contains 220 base pairs of perfect homology (turquoise) and 200 base pairs of interrupted homology (blue and blue/grey, corresponding to Reference (Ref) and Test segments, respectively). Only the Test segment is allowed to vary between constructs.b, Mutation profiles for situations of perfect homology (Test = Ref ) and random homology (Test = GFP). Each mutation profile summarizes C/T and G/A transitions found over the entire sequenced region in 30 spores obtained from a single cross.c, The number of mutations over the total sequenced region for repeat constructs carrying a region of heterology in the middle of the 200-bp test sequence that was otherwise identical to the reference. A[19], a 19-bp dA:dT tract; N[19], a 19-bp tract of random sequence; CA[11], CpA dinucleotide repeated 11 times.d, Schematic representation of tested partial homologies with the matching periodicity of 11 bp: blue - matched bases, grey - random mismatched bases.e, The number of mutations in the total sequenced (magenta) and the invariant (green) regions corresponding to Test segments in c. Regression analysis excludes 0H-11N. r represents Pearson's correlation coefficient. The number of analyzed crosses and spores is provided in .f, Statistical analysis of critical partial homologies. Mutation count distributions are compared by the two-sided Kolmogorov-Smirnov test: *** P < 0.001; NS P ≥ 0.05.The number of analyzed crosses and spores is provided in . Error bars represent s.e.m.

Short periodic tracts of homology must be arrayed with the matching periodicity

a, Ref/Test repeat pairs with 3-bp tracts of homology arrayed with matching periodicities of 10, 11, 12 and 13 bp.b, Representative mutation profiles of repeat constructs in e.c, The number of mutations for repeat constructs in e.d, Repeat pairs with 3-bp tracts of homology arrayed with non-matching periodicities of 11 versus 9, 11 versus 12 and 11 versus 13 bp as compared to the original (matching) periodicity of 11 versus 11 bp.e, Representative mutation profiles of repeat constructs in h.f, The number of mutations for repeat constructs in h.Data represent the mean number of mutations (per spore) over the total sequenced region. The number of analyzed crosses and spores is provided in . Error bars represent s.e.m. Mutation count distributions were compared by the two-sided Kolmogorov-Smirnov test: *** P < 0.001; NS P ≥ 0.05. Each mutation profile summarizes all RIP mutations found over the entire sequenced region in 30 spores obtained from a single cross.

RIP mutation is proportional to the number of triplet frames and does not require MEI3 (RAD51)

a, (i-iv) For each indicated Test segment (red, green, or blue), all potential frames for aligning Test and Ref segments by triplets with the matching periodicity of 11 bp are given. The total number of such frames and overall homology between Test and Ref segments are indicated. Lines (i) and (ii) (red): two partial homologies analyzed in , d. Line (iii) (green): a Test segment that contains as much overall homology as (i) and as many triplet frames as (ii). Line (iv) (blue): a Test segment that provides no triplet frames, but contains more overall homology than (ii) and nearly as much overall homology as (i).b, The mean number of mutations over the total sequenced region for constructs in (blue, red, and green) plotted against the amount of overall homology (left panel) or the number of triplet frames as defined in a (right panel). r represents Pearson's correlation coefficient. Correlation coefficients for the mean and the median values were computed separately.c, Strategy for examining the role of MEI3 in RIP (Methods). Strains SR0 and SR1 were produced from a cross between strains #12433 (Δmei-3) and #12440 (Δspo11) which, along with reference wild-type strain #2489 were obtained from the Fungal Genetics Stock Center.d, Strains used in c were phenotyped and genotyped by PCR. PCR bands correspond to wild-type alleles or the Dp::BAR duplication. The Δmei-3 genotype is verified by the histidine sensitivity assay.e, Mutation profiles summarize 10 spores analyzed from each cross. f, The mean number of mutations (per spore) over the total sequenced region for the crosses shown in e. Mutation counts were compared by the two-sided K-S test: NS P ≥ 0.05.Error bars represent s.e.m.

a, Analyzed constructs contain one or more of the following elements positioned, as shown, to either side of a 707-bp or a 303-bp linker region (grey): 337 bp of perfect homology (turquoise) in direct or inverted orientation; 500 bp of partial homology 4H-7N (blue and blue/white) in direct or inverted orientation, either alone or adjacent to 337 bp of perfect homology in inverted orientation, and with or without 22 bp of random sequence inserted between the perfect and partial homology segments.b, The number of mutations within the partial homology repeat (left panel) and the perfect homology repeat (right panel) in the invariant region. For (ii-vii), corresponding mutation profiles shown in d. *** P < 0.001; ** P < 0.01, NS P ≥ 0.05, ns P-value fluctuates around 0.05. Error bars represent s.e.m.c, Mutation density profiles for constructs (v) and (vii) reveal the effect of the 22-bp insertion at junction between perfect and imperfect inverted repeat units. The invariant region (1000 bp) was divided into sequential non-overlapping 50-bp bins. The number of mutations for each 50-bp bin is plotted. Mutations from two replica crosses for each construct were analyzed together.d, Mutation profiles for repeat constructs ii-vii. Each profile is based on 24 spores (ii, iii, v, vi and vii) or 30 spores (iv) from one representative cross.